Fig 1.
The previously reported carbohydrate binding domain of the lectin does not bind to Gal/GalNAc-containing carbohydrates.
(A) The domain architecture of HgL and the domain boundaries of expressed constructs. (B) Close up of the HgL_03 structure (residues 808-992), highlighting the four modules as insets and showing the locations of the disulphide bonds in yellow. (C) Electron density difference map for HgL_03 in the presence of Gal, GalNAc or LacNAc after subtraction of the density in the absence of Gal, GalNAc or LacNAc, respectively, contoured at 2.0. Positive difference density is coloured pale blue. (D) STD-NMR showing no evidence of binding of different fragments of HgL to Gal. The scale for the reference spectrum has been adjusted 128-fold relative to those of the lower intensity STD NMR spectra. Jacalin is included as a positive control and no binding is observed to HgL_03. The scale for the reference spectrum has been adjusted 128-fold relative to those of the lower intensity STD NMR spectra.
Fig 2.
Structure of the core of the Gal/GalNAc lectin.
(A) A schematic showing domain boundaries for the HgL and LgL chains, derived from the structure. (B) Cryo-EM-derived volume for the lectin complex, coloured as (A). (C) Cartoon representation of the static core of the lectin, with dashed libes used to delineate the light and heavy chains. (D) Ribbon representation of LgL. (E) Comparison of LgL (left) and another β-trefoil protein from Entamoeba histolytica (right, PDB code 6IFA) after structural alignment. The characteristic β-strands and the QxF/QxW motifs are highlighted in purple and orange, respectively.
Fig 3.
LgL contains a Gal-binding domain.
(A) Saturation transfer NMR studies of the binding of galactose (Gal) to native HgL-LgL heterodimer (Lectin) as well as to the heavy chain ectodomain. The scale for the reference spectrum has been adjusted 16-fold relative to those of the lower intensity STD NMR spectra. (B) Comparison of the native HgL-LgL dimer (left) with recombinant HgL (right) as visualised using cryo-EM. The top panels show 2D class averages while the central and lower panels show two equivalent views of 3D volumes. (C) Cryo-EM-derived volume for the LacNAc-soaked lectin. This is coloured as in Fig 2, with LacNAc coloured pink and its location highlighted with a pink box. (D) Electron densities focused onto the Gal/GalNAc-binding site of LgL for apo (left), Gal-soaked (central) and LacNAc-soaked (right) native lectin dimer. Coulomb densities are 0.46 for apo, 0.47 for LacNAc-soaked and 0.9 for Gal-soaked. (E) Models showing close-ups on the carbohydrate binding pocket and the residues involved in interactions with Gal (central) and LacNAc (right and left).
Fig 4.
The C-terminal part of HgL forms a dynamic arm.
(A) Domain boundaries for the HgL and LgL chains, derived from the structures obtained after 3D classification of the C-terminal half of HgL. (B) Three different conformations of the dynamic extension of the lectin, revealed after 3D classification of cryo-EM data. The colours are as in (A). The galactose binding site is labelled with a lime green circle. (C) Overlay of the three conformations. The paths taken by the arm are highlighted by the lines, with a two-sided arrow indicating the likely motion to transition between conformations. The globular core is coloured in grey, the arm is coloured as in (A). (D) A close-up of the structures of mode 1 (left) and mode 3 (right), focusing on the junction between arc domain 7 and domain 8. The black arrows highlight the two different directions followed by the polypeptide backbone at this hinge-point.